L.11 Cytochrome C Comparison Lab

PROTEINS AND EVOLUTION

Genes are made of DNA and are inherited from parent to offspring. Some DNA sequences code for mRNA which, in turn, codes for the amino acid sequence of proteins. Over time, random mutations in the DNA sequence occur. As a result, the amino acid sequence of Cytochrome C also changes. Cells without usable Cytochrome C are unlikely to survive. The cytochrome C is a small protein found loosely associated with the inner membrane of the mitochondrion. It is found in eucariotic cells and has an hemeprotein. It is essential to the electrone transport chain and it is involved in using energy in the cell (ATP).

The purpose of this practice is to compare the relatedness between organisms by examining the amino acid sequence in the protein.

METHOD

First, we compared the amino acid sequence of Cytochrome C in various organisms:
-horse, donkey, whale (mammals)
-penguin, chicken (birds)
-snake (reptile)
-moth (insect)
-wheat (plant)
-yeast (fungi)

1- We marked the amino acids which were different.
2-Then we counted and recorded the total number of differences.
3- We shared the data with the rest of the class to complete Table 1.


After that, we made a cladogram (branching tree)
1- The two most closely related species had the fewest differences in amino acid sequence. We placed the two most closely related species on the two shortest branches of the tree.
2-Then we placed the next two closest species on the next shortest branches.
3- And we continued until all the species had been placed.

RESULTS AND OBSERVATIONS

CONCLUSIONS

There are 0 differences between chickens and turkeys in their Cytochrome C amino acid sequence.

We think that the horse and the zebra have 1 or 2 differences, like the donkey and zebra.
To make this prediction we used this information: if they can reproduce and if the offspring will be fertile or not, we compared organs, compared embrios...

More closely related organisms have more similar Cytochrome C because evolutionarily, it hasn't been that long since they separated. If the species are close, it means that less time has passed since they separated (there are less accumulated mutations). They have a common ancestor.

Other data, including genes, suggests that fungi are more closely related to animals than plants. But Cytochrome C data suggests that fungi, plants and animals are equally distantly related because if there are more than 40 genes, there are too many mutations to see it clearly.

L9. Protein identification

On december 22nd we did an experiment about proteins. The objectives are to identify the peptide bonds and to compare the protein concentration in different foods.

Biuret's test
The Biuret's test is a chemical test used for detecting the presence of peptide bonds (the bonds that link together amino acids that form chains --> proteins). A peptide bond can be broken by hydrolysis. The intensity of the colour of the Biuret's test is directly proportional to the protein concentration. The solution to be tested in treated with a strong base followed by a few drop sof copper II sulphate. If the solution turns purple protein is present. Only peptides with a chain of at least 3 amino acids give a significant measurable colour shift with these reagents (polypeptides).


MATERIALS

-250 ml beakers
-Test tube rack
-6 test tubes
-6 x 10 ml pipet
-Mortar
-Glass marking pen
-Gloves
-Goggles
-Milk
-Rice milk
-Egg (white and yolk)
-Yogurt
-Potato
-Distilled water
-NaOH 20%
-10 drops of CuSO4

PROCEDURE

First of all we had to dilute the protein:
1- We added 100 ml of distilled water to each 250 mL beaker, and with the glass marking pen we labeled them as M (milk), R (rice milk), EW (egg white, EY (egg yolk), Y (yogurt) and P (potato).
2- Then we added 10 ml of a dispersion of each food in the belonging beakers, we separated the egg white and yolk in two different beakers and mashed the potato.

Then we prepared the samples:
3- We cleaned and dried the test tubes and labeled them like the beakers. Each group used the dispersions from the same beakers. Then we added 2 mL of every food dilution of the beaker to each corresponding test tube.
4- After that we added 2 mL of 20%NaOH dissolution to each test tube, Andrea did this because NaOH is caustic so she had to put gloves and goggles on.
5-We shook gently and added 5 drops of CuSO4 in each test tube. Then we allowed the mixture to stand for 5 minutes.
6-Finally we saw the colour change (to pink or purple) amd we compared the test tubes.

Results and observations:
Milk - positive
Rice milk - negative
Egg white - positive
Egg yolk - negative
Yogurt - positive (it has less water, the protein is more concentrated)
Potato - positive, but our teacher said that it had to be negative because it had starch.

We saw that all the foods of animal origin gave a positive result, they all have proteins. Rice milk is negative because it has starch like the potato (but strangely the potato was positive). Also, we ordered the food from more concentration of protein to less according to the colour: 1- egg white, 2- yogurt and potato, 3- milk.

QUESTIONS

1- Which food has proteins?
Eggwhite, milk yogurt and potato.

2- Which food has more proteins? Why?
Eggwhite because it's animal food and it's rich in proteins. Then the yogurt and potato and finally the milk.

3-Do you find any difference between rice milk and cow milk?
Rice milk doesn't have proteins however cow milk does as i had said before.

4- Is there any difference among milk and yogurt? why?
Yes, yogurt has more protein than milk because the protein is more concentrated than in the milk, it has less water.

L8. Saponification

On the 1st of december we decided to experiment the saponofication, we wanted to make soap. Saponification is a reaction that produces soap when you mix fats and a strong base (NaOH).

MATERIALS

-32 gr NaOH
-90 ml H2O
-270ml of olive oil
-safety goggles
-2 beakers
-gloves
-balance
-stirring rod
-spatula
-heating plate

PROCEDURE


First we put on our safety goggles and gloves to protect ourselves. Then we mixed in a 600 ml beaker the 32 grams NaOh with the 90 ml of water, slowly, we noticed that the beaker heated up, it is a endotermic reaction! After that we slowly started to pour the olive oil in the beaker, at the same time mixing the mixture with the stirring rod. We mixed it for 20 minutes, also we heated the beaker on the heating plate to accelerate the reaction. Then we put a couple of drops of aroma and finally we put the soap mixture in a mold.

Results and observations: We will let the soap mixture sit for a couple of days and then we will obtain hard soap. The reaction of saponifiaction:

First by hydrolisis we obtained glicerine and oleic acid, then the oleid acid joined the NaOH and soap and water were formed.

L7. Lipids properties

On december 1st we did a new interesting experiment related with lipids. These compounds are characterized as natural substances that do not mix with water but dissolve in organic solvents, C, H and O are the principal elements of lipids. The objectives of this experiments are to test the solubility of lipids, indetify lipids in liquids compunds and to understand what is an emulsion and the effect of detergents. We can divide this experiment in three.

MATERIALS

-Test tube rack
-250 mL beaker
-Water
-6 test tubes
-Cellulose paper
-Dropper
-Scissors
-Glass rod

-Olive oil
-Soap
-Milk with different fat content (full-cream, semi-skimmed, and skimmed milk)
-Petroleum ether
-Ethanol
-Sudan III

SOLUBILITY OF SOME LIPIDS
PROCEDURE

 1-First we cleaned and dried three test tubes, we labeled them as W (water), E (ethanol, and PE (ether).
2- Then we added 3 drops of oleic acid to the three test tubes.
3- We added 1 ml of water in the first test tube (W).
4- 1 ml of ethanol in the second test tube (E).
5- 1ml of petroleum ether in the thirs test tube (PE).
6- Finally we shaked each test tube and recorded solubility and observations in our worksheet.

Results and observations: We saw that in the test tube W the oleic acid didn't dissolve, in the E test tube micelles had formed and in the last PE test tube it dissolved completely because ether is an organic dissolvent.

LIPIDS IDENTIFICATION:
A. TRANLUSCENT MARK:

1- Our teacher gave us two pieces of cellulose paper.
2- In the first paper we put one drop of water, we saw a translucent spot and then we waited a little bit to see what was happening.
3- Then we put a drop of olive oil on the second piece of cellulose paper and we saw a translucent spor also. We waited to see what was happening.
4- Finally we wrote the results in our worksheet.

Results and observations:  The drop of water of the first piece of paper dried after a while and didn't leave any marks but the drop of oil was still visible when dried, it was translucid.

B. SUDAN III dye

1-We took the W test tube of the first experiment and added 2 drops of Sudan III.
2- Then we prepared three test tubes with full-cream, semi-skimmed and skimmed milk and also put 2 drops of Sudan III in each test tube to stain it.

Results and observations: The water had to turn black if there were fatty acid in the solution but in our experiment it didn't work and nothing happened.

PERMANENT EMULSION:

1- We took a 250 ml beaker and put 100 ml of water there.
2- Then we added 1 ml of olive oil and with a glass rodd we stirred the mixture vigorously and let it stand for a few minutes.
3- After taking note of what was happening we added 2 drops of soap and stirred the mixture again, and waited for a little bit to notice the difference.

Results and observations: First when we added oil to the water a layer of oil formed in the top part of the water. When we added soap microdrops appeared, little micelles. Soap doesn't dissolve the lipid, it just wraps it so the oil layer can't be formed.

QUESTIONS:
1-From your observations, which compunds can dissolve lipids?
Organic solvents can dissolve lipids, for example in our case ether dissolved them.

2-Do the oil and water mix? No
What can you conclude about the polarity of oil if you know water is polar?
I can say that lipids tend to be non-polar because they are made of longs chains of hydrocarbons with relatively little oxygen (hydrocabron chain), and this fatty acid tail is hydrophobic.Water molecules are strongly attracted to each other, this is the same for oil, because they are more attracted to their own molecules they just don't mix together. They separate and the oil floats above the water because it has a lower density.

3- Why is olive oil liquid at room temperature? And why not the lard?
Olive oil is liquid at room temperature because it is unsarurated (the chain bends and it only bonds on one part, it's easier to break). Lard isn't liquid because it's saturated (so it's harder to break the bonds)

4-Why does a lipid leave a translucent spot on paper?
In their liquid state oils and fats easily penetrate into the pores of dry substances. The lipids soak into the paper fibres, but the lipid spot evaporates much more slowly than water (because the forces that hold the lipid molecules together are stronger than those in water molecules)

5-  Which type of milk contains more lipids? Why?
The full-cream milk contains more lipids because it has more fat (fatty acids).

6- Did the oil and water mix when you added soap? 
Yes. Detergent is attracted to both water and oil helping them all join together and form something called an emulsion.

7-What did the soap do to the fat? 
Soap on one end it is hydrophillic (that is polar) and on the other end it is hydrophobic (non polar). So, the polar end of the soap molecule will attract the water molecules while the non-polar end of the molecule attracts the oil molecules.
Soap doesn't dissolve the lipid, it just wraps it so the oil layer can't be formed.

8- Can you think about process and locations were compounds like the soap would be important to an animal? 
Soap makes insoluble particles become soluble which allows water to rinse the particles away. This means that while oil doesn’t naturally mix with water, soap can suspend oil/dirt in such a way that it can be removed. They are useful in bile acids.

L6. Fehling's Test: Reducing sugars

On Monday 27th of October after doing our last experiment we had a little bit of time left so we decided to do another experiment realted with the sacharides. Fehling's solution is a chemical test used to differentiate between reducing and non-reducing sugars. This test is based on the reaction of a functional group of sugar molecules with Fehling's regent. The objectives were to identify reducing sugars, comprehend redox reactions and understand the relation between structure and reducing ability of some sugars.

MATERIALS:
-Test tube rack
-10 mL pipet
-Distilled water
-5 test tubes
-Spatula

-Lactose
-Maltose
-Sucrose
-Glucose
-Starch
-Fehling's A and B solutions

PROCEDURE:
1-We took 5 test tubes and labeled them: G, M, S, L, SU
2-We put 2 mL of distilled water inside each tube.
3- Then, with differents spatulas we put a small amount of each sugar. 
4-After that we added 2 mL of Fehling's A solution and then Fehling's B.
5-We placed each test tube in a boiling water bath (250 mL beaker on a hotplate stirrer)
6- Then we observed what was happening.

Results, observations and conclusions:  Glucose, maltose and lactose have reducing ability because they turned from deep blue to a red colour.  However, sucrose, in which the anomeric carbons of the two units are linked together, is a non-reducing disaccharide since neither of the rings is capable of opening. Starch is not a reducing sugar either, because the first ring cannot open up, there's no hydrogen on the circled oxygen to allow for ring opening. Similarly the next ring, and the next ring, et cetera, cannot open up.

QUESTIONS

1-From your observations and the structures of the sugars given above, indicate which functional group in the sugar molecules reacts with Fehling's reagent.

The OH group is the one that reacts with Fehling's reagent because when it is free the sacharide will have the reducing power.

If the bond is monocarbonilic the sacharide will have reducing power, that's why all monosacharides have it (the OH from the C1 is always free).

 

2-Compare the results you obtained for the Fehling's test of starch and Fehling's test of hydrolyzed starch. Explain your results.

I haven't done the Fehling's test of hydrolized starch but i can deduce it:

In the Fehling's experiment the starch doesn't have a reducing power because the OH is not free but when the starch is hydrolyzed it turns into glucose and glucose has a free OH because it's a monosacharide. Also the last glucose of the starch chain will have a reducing power (the ones that are in the ends).

The starch components are alfa D glucoses: amylose alfa (1 -> 4) linear chain, and amylopectine alfa (1->6) ramifications.

 

3-Would have you obtained a Fehling's positive test if you had hydrolyzed the sucrose? Why? 

Yes i would. Hydro meaning "water", and lysis, meaning "separation" usually means the cleavage of chemical bonds by the addition of water. So this means if we hydrolyze sucrose we will obtain glucose and fructose and they both will have a free OH because they are monosacharides. 

4-What does "reducing sugars" mean? 

A reducing sugar is the one that reacts positive to the Fehling's test. This means that they are capable of reducing coper II ions to copeer I ions. When the sugar to be tested is added to the Fehling's solution and the mixture is heated, some sugars can be oxidized (to lose electrons) and the Fehling's mixture can obtain the electrons (reduced). 

 

L5. Saccharides properties

On Monday 27th of October we did our weekly experiment in the laboratory. Saccharides are organic molecules consting of C, H and O atoms. The main objectives of this experiment were to identify the different sugars from its properties, differentiate mono and disaccharides and understand the relation between structure and some properties.

MATERIALS:
-Test tube track
-19 mL pipet
-Water
-5 test tubes
-1 dropper
-A spatula

-Lactose
-Maltose
-Glucose
-Sucrose
-Starch
-Lugol's iodine

PROCEDURE: 

In the first part of the experiment we had to test some physical properties of the saccharides we had in the lab: flavour, structure and colour. To know the flavour we put a small amount of each saccharide in our hand and tasted it. To see if they had crystals or not we observed a small amount of each saccharide on a clock glass under magnification. And to determine the colour we chose between white, transparent or creamy. 

Then we proceed to do the next part of the experiment, the test of solubility. 
1. We cleaned and dried 5 test tubes and labeled them "G, M, L, SU, S"
2. Then we put 5 mL of water in each test tube.
3. With the aid of a spatula, we put a small amount of each saccharide inside the labelled test tube and observed if they were soluble or insoluble. 

Lugol's iodine test
To each test tube we added two drops of Lugol's iodine (it acts as a starch detector) and tested if the reaction was positive or negative. Lugol's is a solution of elemental iodine (I) and potassium iodine (KI) in water that is used to test a saccharide. The reaction is positive when iodine reacts by turning from yellow to a purple, dark-blue colour. 

Results, observations and conclusions:
Lastly we completed our chart with the information. 

QUESTIONS:

1-Write the empirical formula of each saccharide that you have used. Show structures of the five saccharides. Classify each one in one group: mono, oligo, or polysaccharide.

Glucose: monosaccharide

 

C₆H₁₂O₆

Maltose: disaccharide

 C₁₂H₂₂O_{11  ·    H2O}     



                  

Sucrose: disaccharide 

C₁₂H₂₂O₁₁





Lactose: disaccharide

C₁₂H₁₂O_{11  ·}    H₂O 

Starch: polysaccharide

C₆H₁₂O₅ 








2-Which of the monosaccharides are aldoses and which are ketoses? 
Glucose: aldose // Maltose: aldose // Sucrose: ketose // Lactose: aldose // Starch: aldose

3-Which bond links monosaccharides?
O-glycosidic bond.

4-Which saccharides are sweet? Is this property related to the structure og the molecule?
Glucose, maltose and sucrose are sweet. Polysaccharides aren't sweet because of the size of the molecule, the human tongue can detect the short one- and two-molecules of the short chains. The long chains just slide on by.
 
5- Which saccharides are insoluble? Is this property related to the structure og the molecule?
 Starch because it's a big molecule (polysaccharide) with big molecular weight. 

6-Which saccharide has reacted with Lugol's iodine solution? 
 Starch.

7-Which kind of foods contain starch? 
Carbohydrates, which include pastas, breads, rice, cereals, flour, and beans. Starchy vegetables, such as potatoes, peas and corn.

8- Calculate the energy that comes from the nutrition facts label from a cereal:  
Calculate the energy that comes from the saccharides.

1 g of saccharides ----------> 4,2 Kcal
23 g of saccharides ---------> x

23 · 4,2 / 1 = 96,6 Kcal

L4. pH

On Monday 13th of October we started another experiment called "pH" the main objectives were to measure different pH values of organic and inorganic solutions and to prove different methods of measuring pH. The pH is the measure of the acidity or basicity of a solution. Solutions with pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline. Pure water has a pH close to 7, it's neutral. The pH is defined as the following equation:

pH = -log (H+)

MATERIALS:
-Distilled water
-Milk
-Wine
-Tomato
-Coffee
-10% NaOH solution
-10% HCl solution
-NH3 solution
-Soap Solution
-Bleach
-Universal indicator paper strips
-pH-meter
-Acetic acid
-Tongs
-Lemon
-Beakers
-Clock glass
-5 test tubes
-Test tube track
-10 mL pipet
-Funnel
-Graduated cylinder

PROCEDURE:

Universal indicator paper strips experiment:
With this experiment we wanted to measure the pH of different solutions, so we put different solutions in small beakers of 250 mL. We used: milk, wine, tomato, coffee, soap soluton, NH3, NaOH and HCl. For example: we squeezed the tomato in a clock glass and then we put the piece of paper  into the solution. Then we removed the indicator paper and compared its colour with the colour chart.

Results, observations and conclusions: This picture shows the results of each solution, it shows its pH.

"How does concentration affect pH?" experiment:
First we squeezed the lemon juice inside a graduated cylinder using a filter made of celluose paper and a funnel, then we prepared a test tub rack with 5 test tubes cleaned with distilled water; we marked the tubes with the label: A, A1, A2, A3 and B. The next step was to add 10 ml of lemon juice to tubes A and B, then we took the A tube and put 5 mL of its lemon juice to test tube A1. Then 2,5 ml of the test tube A1 to tube A2. Lastly, we took the A2 tube and put 1,2 mL of it lemon juice to tube A3. The final step was to add distilled water to each test tube until it had the same volume as test tube B (10mL). Then we measured each pH with the strips and with a pH-meter.

Results, observations and conclusions: 
We calculated the concentration by doing this equation:

Concentration (%) = (Volume of Juice / Total Volume) · 100

Our hypothesis is that if we have a high lemon juice concentration we expect it to be the most acid because it has the most concentration of protons. But in the chart we see that in our case this is not happening; the test tubes with a higher concentration of lemon juice in them, have a lower pH, they are the most acid!
Maybe the problem was that the pH-meter wasn't calibrated correctly.
Our classmates Ignacio and Eduard got it right because they were the first ones to use the pH-meter, and we can accept our hypothesis. You can see how they did it HERE.

Graphic:

QUESTIONS:

1-Which of the solutions fave an acid pH? HCl, vinegar, wine, tomato, coffee and milk.

2-Which of the solutions were alkaline? Soap, bleach and NaOH.

3- Which of the solutions were neutral? Did you expect this results? Explain 
Distilled water was neutral, and yes we expected this results because it has no mineral salts and it has the same number of H+ and OH- ions.

4- How does a pH of 3 differ from pH 4 in terms of H+ concentrations? A pH of 3 means that it has 10^-3 H+ and is more acid and a pH of 4 has 10^-4 H+ .

5-In the second part of the experiment, you have compared the pH of the same product (lemon juice) in different concentrations. In this case explain:
a) Which is the dependent variable? pH
b) Which is the independent variable? The concentration
c) Which if the problem that we want to solve? We want to see if the pH depends of the concentration.

6-Which pH do you think that gastric juices might have? Why? Do you think that intestinal pH has the same pH why?
They will have 2 because gastric juices have HCl.  In the small intestines, the duodenum provides critical pH balancing to activate digestive enzymes. The liver secretes bile into the duodenum to neutralize the acidic conditions from the stomach, and the pancreatic duct empties into the duodenum, adding bicarbonate to neutralize the acidic chyme, thus creating a neutral environment. The mucosal tissue of the small intestines is alkaline with a pH of about 8.5

7-Which pH do you think that blood might have? Why? Neutral because the intern fluids have to have a neutral pH because if it wasn't like that we would die.

8-What is acid rain? which are the consequences in the ecosystems and how is its formation pattern? Is rain in Barcelona acid or alkaline?
Acid rain is rainfall made so acidic by atmospheric pollution that it causes environmental harm, chiefly to forests and lakes. The main cause is the industrial burning of coal and other fossil fuels, the waste gases from which contain sulphur and nitrogen oxides which combine with atmospheric water to form acids. In Barcelona the rain is alkaline, maybe just slightly acid.